Reduced commissural connectivity of the habenula in depression: Translational evidence from human DTI and rodent ultrastructural analyses

Reduced commissural connectivity of the habenula in depression: Translational evidence from human DTI and rodent ultrastructural analyses | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Reduced commissural connectivity of the habenula in depression: Translational evidence from human DTI and rodent ultrastructural analyses Hyun Woo Lee, Esther Yang, Hyeijung Yoo, WS Tae, Soo Hyun Yang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8207931/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Dysfunction of the habenula is increasingly implicated in the pathophysiology of major depressive disorder (MDD), yet the contribution of the habenular commissure (Hbc)−the fiber tract connecting the bilateral habenulae–remains largely unexplored. Here, we combined in vivo human neuroimaging and preclinical ultrastructural analyses to identify a previously unrecognized microstructural signature of MDD. Using diffusion tensor imaging (DTI), we observed robust reductions in fractional anisotropy (FA) and axial diffusivity (AD) within the Hbc of individuals with MDD compared with healthy controls. Notably, FA and AD values were inversely correlated with Hamilton Depression Rating Scale (HDRS) scores, linking Hbc integrity to clinical symptom severity. Complementary electron microscopy of a chronic social defeat stress model revealed reduced axon diameter and myelin thickness of interhabenular connectivity, providing convergent evidence for commissural atrophy. These cross-species data identify the habenular commissure as a novel locus of microstructural pathology in depression and highlight its potential as a diagnostic and pathophysiological biomarker for MDD. Health sciences/Diseases/Psychiatric disorders/Depression Biological sciences/Neuroscience Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Major depressive disorder (MDD) is a prevalent psychiatric condition, affecting approximately 15−18% of individuals over their lifetime and is a leading contributor to global disease burden 1 , 2 . Core features of MDD include deficits in motivation and reward processing, and anhedonia is frequently observed in affected individuals 3 , 4 . Neurobiological evidence implicates dysfunction of the dopaminergic mesocorticolimbic pathway, which originates in the ventral tegmental area (VTA) and projects to regions including the prefrontal cortex, anterior cingulate cortex, and ventral striatum 5 , 6 . Neuroimaging studies have consistently identified structural and functional abnormalities within these circuits in MDD 7 – 9 . Recent work highlights the lateral habenula (LHb) as a critical regulator of midbrain dopaminergic and serotonergic centers. Hyperactivity of the LHb has been observed in animal models of depression, where it inhibits VTA dopamine neurons, potentially contributing to anhedonia-like behaviors 10 – 12 . Despite increasing interest in the habenula, its commissural connection–the habenular commissure (Hbc), a small white matter tract interconnecting the bilateral habenulae 13 –remains largely unexplored in the context of MDD. Given that the LHb exerts bilateral control over the monoaminergic system, the integrity of the Hbc may represent a critical but overlooked factor in depressive pathophysiology. Diffusion tensor imaging (DTI) allows non-invasive assessment of white matter microstructure and has revealed alterations in major tracts in MDD, including the corpus callosum 14 – 18 . However, the Hbc has never been investigated in clinical populations, and thus its microstructural properties remain entirely uncharacterized. We focused on four standard DTI indices: Fractional anisotropy (FA) captures overall microstructural organization; axial diffusivity (AD) is particularly sensitive to axonal injury or reduced axonal caliber; radial diffusivity (RD) has been associated with myelin status; mean diffusivity (MD) reflects overall water mobility within tissue 19 . In this study, we examined whether the Hbc exhibits structural alterations in individuals with MDD. Using DTI, we quantified Hbc microstructure with standard indices of axona and myelin integrity. To further enhance microstructural resolution beyond the limits of clinical imaging, we complemented human DTI analyses with electron microscopy (EM) in a chronic social defeat stress (CSDS) mouse model. Through this integrative human-preclinical approach, we sought to determine whether Hbc microstructural abnormalities are associated with MDD and to evaluate their potential as a novel neuroimaging biomarker. Methods Ethics statement The procedures for animal experiments adhered to the guidelines of Korea University and were approved by the Korea University Institutional Animal Care and Use Committee (study approval number KOREA-2021-0073). Regarding the human neuroimaging study, the study protocol was approved by the Institutional Review Board (IRB) of Korea University Anam Hospital (2018AN0118). Written informed consent was obtained from all participants prior to their involvement in this study. The study methodology adheres to the approved guidelines and the principles outlined in the Declaration of Helsinki. Participants A comprehensive cohort of 43 individuals diagnosed with MDD, comprising 31 females and 12 males, along with 25 healthy control participants, including 16 females and 9 males, was enrolled in the human brain imaging analysis. Recruitment of MDD patients occurred between July 2018 and March 2021 at the outpatient psychiatric clinic of Korea University Anam Hospital, Seoul, Republic of Korea. Inclusion criteria for MDD patients encompassed adults aged 19–64 years, and the diagnosis was confirmed using the Structured Clinical Interview for the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) 20 , conducted by two experienced board-certified psychiatrists (K.-M. Han and B.-J. Ham). Exclusion criteria included comorbid major psychiatric disorders (including personality and substance use disorders), MDD with psychotic features, acute suicidal or homicidal tendencies requiring inpatient treatment, history of serious or unstable medical illness, primary neurological conditions (e.g., cerebrovascular disease, Parkinson's disease, or epilepsy), recent abnormal results on physical examination or laboratory tests, pregnancy or nursing status, and any contraindications for magnetic resonance imaging (MRI), including claustrophobia. Twenty-five healthy control participants aged 19–64 years were recruited from the community through advertisement, following the same exclusion criteria that were applied to the MDD group by two psychiatrists. Healthy controls were included only if two board-certified psychiatrists confirmed the absence of any psychiatric history. Sociodemographic and clinical characteristics were systematically collected from both groups. The severity of depressive symptoms in all participants was evaluated by two psychiatrists using the 21-item Hamilton Depression Rating Scale (HDRS) at the time of the MRI scan 21 . The duration of illness was assessed as the cumulative number of months with depressive episode(s) throughout the individual’s lifetime, employing life-chart methodology. Among the 43 MDD patients, 38 were taking psychotropic medication, including antidepressants or antipsychotics, at the time of study enrollment. Detailed information regarding psychotropic medications is presented in Table 1 . Table 1 Demographic and clinical characteristics for the participants Characteristics MDD (n = 43) HC (n = 25) P -value (t, c 2 ) Age 35.37 ± 13.72 37.48 ± 13.56 0.542 (t = 0.613) Sex (Female/Male) 31 / 12 16 / 9 0.486 (chi 2 = 0.485) Education years 13.98 ± 2.69 16.80 ± 1.96 < 0.001 (t = 4.589) HDRS-21 score 18.95 ± 5.75 0.52 ± 0.82 < 0.001 (t = −20.667) Illness duration (months) 28.19 ± 20.11 NA NA Drug-naive / Drug-treated patients (n) 4 / 39 NA NA Medication, n Antidepressants SSRI 15 NA NA SNRI 13 NDRI 1 NaSSA 1 Etc 1 Combination of AD 8 Antipsychotics None 21 AP 13 Combination of AP 5 Data are presented as mean ± standard deviation for age, years of education, HDRS-21 score, and illness duration. P -values for sex distribution were obtained using the chi-square test. P -values for comparisons of age, years of education, and HDRS-21 scores were obtained using an independent t -test. HCs, healthy controls; MDD, major depressive disorder; HDRS-21, 21-item Hamilton Depression Rating Scale; SSRI, selective serotonin reuptake inhibitor; NDRI, norepinephrine-dopamine reuptake inhibitor; SNRI, serotonin-norepinephrine reuptake inhibitor; NaSSA, noradrenergic and specific serotonergic antidepressant; Combination of AD, combination therapy with two or more antidepressants; APs, antipsychotics; Combination of AP, combination therapy with two or more antipsychotics. MRI acquisition Participants underwent MRI-scanning at Korea University Anam Hospital using a 3.0 Tesla Siemens MR scanner (Magnetom Prisma, Siemens Medical Solutions, Erlangen, Germany) equipped with a 64-channel head and neck coil. A coronal 3D T1-weighted magnetization prepared rapid gradient echo (MPRAGE) sequence was acquired perpendicular to the long axis of the anterior commissure to the posterior commissure in the midsagittal plane, employing the following parameters: slice thickness (ST) = 1.0 mm, no gap, number of slices = 384, TR/TE = 2300/2.26 ms, flip angle = 9°, number of signal averages = 1, matrix = 256 × 256, and field of view (FOV) = 256 mm × 256 mm. The acquisition of DTI data utilized a single-shot spin-echo planar imaging pulse sequence with the following specifications: number of diffusion gradient directions = 64, matrix = 112 × 112, FOV = 224 × 224 mm 2 , slice thickness = 2.0 mm, voxel size = 2.0 × 2.0 × 2.0 mm 3 , echo time = 55 ms, repetition time = 6100 ms, b-value = 1000 s/mm 2 , and flip angle = 90°. Diffusion MRI preprocessing Diffusion preprocessing was performed using the FMRIB Software Library (FSL) 22 . Because the diffusion acquisition did not include reverse phase-encoding images, susceptibility-induced distortion correction using TOPUP could not be applied. Geometric distortions were minimized by using a relatively short echo time (TE = 55 ms) and a posterior–anterior phase-encoding direction, reducing susceptibility-related warping in deep midline structures such as the habenular commissure (Hbc). Eddy current and head-motion artifacts were corrected using the eddy_cuda implementation, which also performs slice-wise outlier replacement and within-volume motion correction. All diffusion-weighted volumes were visually inspected to ensure geometric stability in the habenular region. Gradient nonlinearity correction and N4 bias-field correction were subsequently applied. Bilateral habenula regions of interest (ROIs) were manually delineated on each participant’s T1-weighted anatomical scan following established anatomical boundaries 23 , 24 . T1 images were registered to diffusion space using affine transformation, and ROI alignment was visually verified for each participant. Tractography and diffusion metrics Diffusion tensors were reconstructed in DSI Studio 25 using a standard tensor model, yielding voxel-wise FA, MD, RD, and AD maps. To visualize multidirectional fiber orientations within the Hbc, generalized q-sampling imaging (GQI) reconstruction was additionally applied to estimate the orientation distribution function (ODF). Deterministic streamline tractography was conducted in DSI Studio. The left habenula served as the seed region, and the right habenula as the target. A QA-based anisotropy threshold was empirically determined based on prior habenula tractography studies. Tracking was performed with an angular threshold of 30°, a step size of 0.5–1.5 voxels, and angular perturbation of 0–95% to avoid local minima. Streamlines shorter than 5 mm or longer than 15 mm were excluded. For each participant, 100 streamlines were generated, and ODF was computed to characterize local fiber orientations. Animals and chronic social defeat stress model Male C57BL/6J mice (Japan SLC, Inc., Shizuoka, Japan) and ICR mice (KOATECH, Korea) were obtained and housed in a facility under a 12-h light/dark cycle at 21–24°C, with ad libitum access to food and water. The mice underwent a one-week habituation period to the housing conditions before being randomly assigned to either an experimental group or a control group (Naïve). Mice in the experimental group were exposed to chronic social defeat stress (CSDS) through daily 10-min encounters with ICR mice for 10 days, while control mice received no stress exposure and remained in their home cages. On the final day of the stress protocol, mice from both groups were immediately sacrificed following the last exposure. After decapitation, brains were rapidly extracted and processed for volumetric analysis. Animal behavioral paradigms The social interaction test consisted of two 150-s sessions: one with a social target (ICR mouse) and one without, as previously detailed 26 . The test was conducted in an open-field arena (45 cm × 45 cm × 40 cm) containing a transparent box (10 cm × 6.5 cm × 20 cm) positioned in the interaction zone. During the first session, the box was empty, and during the second session, it contained the ICR mouse. Time spent in the interaction and non-interaction zones was quantified using EthoVision XT 12 (Noldus, Wageningen, Netherlands). In the elevated zero maze test, mice were placed in the closed quadrant at the beginning of the session and allowed to explore the maze freely for 5 min. Behavioral parameters, including total distance traveled, time spent in each quadrant, frequency, and latency to enter the open quadrant, were recorded and analyzed using EthoVision XT. The tail-suspension test was performed using a four-chamber apparatus separated by opaque acrylic partitions. Mice were suspended by the tail in each chamber using adhesive tape, and their behavior was recorded for 6 min. Immobility was quantified during the final 4 min of the session. Tissue sampling and preparation Mice were perfused transcardially with 0.9% saline followed by a fixative containing 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). Brains were removed and post-fixed overnight at 4°C in the same fixative. Coronal brain blocks (approximately 1.5 mm thick) containing the habenula commissure (Hbc) were prepared using a mouse brain matrix (Harvard Apparatus). The Hbc was dissected from each block and washed twice with 0.1 M phosphate buffer. It was then post-fixed in 1% osmium tetroxide for 90 min, dehydrated through an ascending ethanol series followed by propylene oxide, and embedded in Epon 812 (Oken Shoji, Japan). Electron microscopic analysis of mouse Hbc For electron microscopy, ultrathin sections (70 nm) were obtained from the middle portion of the Hbc using a Leica EM UC6 ultramicrotome (Leica Microsystems, Wetzlar, Germany). Sections were mounted on 200-mesh copper grids and examined with a Hitachi H-7650 transmission electron microscope (Hitachi, Tokyo, Japan) at 80 kV. Ten images were randomly acquired from selected regions within the Hbc at a magnification of 20,000×. Images were analyzed using ImageJ by an investigator blinded to the experimental groups. Myelinated and unmyelinated axons were manually traced, and ImageJ was used to measure axon and myelinated axon diameters (Feret’s diameter, µm) and areas (µm 2 ). Myelin thickness was defined as half the difference between the myelinated axon diameter and the axon diameter. Axon diameter distributions were characterized using an average of 127 axons per segment per control mouse and 178 axons per segment per CSDS mouse. Statistical analysis For the examination of human DTI data, multivariate analysis of covariance (MANCOVA) and one-way analysis of covariance (ANCOVA) were conducted. The four DTI parameters (FA, MD, RD, and AD) of the Hbc were treated as dependent variables, and groups (MDD vs healthy control) served as the independent variables. Age, sex, and years of education were included as nuisance covariates. In the supplementary analyses, potential correlations between illness burden-related clinical characteristics (severity of depressive symptoms [HDRS total score] and duration of illness) and DTI parameters were assessed using Pearson’s partial correlation. For analyses including the total sample, age, sex, and years of education were included as covariates. For analyses within the MDD group, illness duration and medication status (coded as 0 for drug-naïve patients; 1 for patients taking psychotropic medication) were additionally included. P-values were corrected using Benjamini-Hochberg procedure. To examine sociodemographic and clinical differences between the MDD and healthy control groups, independent t -tests were used for age, years of education, and HDRS scores, and chi-square tests were used for sex distribution. All statistical analyses were performed using R4.3.0. Comparisons of behavioral tests between CSDS and control mice were analyzed using two-tailed unpaired Student’s t -test. Correlations between behavioral and electron microscopy parameters were assessed using Pearson’s r . Graphs were generated using the ggplot2 package. Results Tractography of the habenular commissure Using DSI Studio, we conducted an in-depth exploration of the habenular commissure, generating tractography results to depict the connections between the two habenular nuclei. The brain's 3T MRI scan provided a comprehensive view of both the individual habenular nuclei and the habenular commissure, serving as a reference for the subsequent analyses (Fig. 1 a and b). We closely examined specific ROIs associated with the habenular nuclei, emphasizing their significance in the overall analysis (Fig. 1 c). We also examined the orientation distribution function (ODF) of the habenular commissure, which describes the directionality of multidirectional diffusion in white matter regions. The ODF revealed a distinctive pattern of horizontal, unidirectional diffusion within the habenular commissure, providing insights into the microstructure of this neural pathway. To enhance clarity, red arrows were incorporated into the figure, indicating the direction of the ODF within the voxel of the diffusion tensor imaging (DTI). This comprehensive examination not only highlights the technical prowess of DSI Studio but also deepens our understanding of the habenular commissure's microstructural features, contributing to advancements in the field of neuroanatomical research (Fig. 1 d). Human DTI results Table 1 summarizes demographic and clinical characteristics, including age, sex, years of education, HDRS score, illness duration, and psychotropic medications (i.e., antidepressants and antipsychotics). Age and sex did not differ between the groups ( p > 0.1), but the MDD group had fewer years of education than healthy controls ( t = 4.589, p < 0.001, Table 1 ). Multivariate analysis of covariance revealed a significant main effect of group on Hbc DTI indices (Wilks’ λ = 0.670, Pillai’s trace = 0.329, F (4,60) = 7.36, p < 0.001). Patients with MDD showed significantly reduced FA (F (1, 63) = 6.997, adjusted p = 0.023, partial η² = 0.129) and AD (F (1, 63) = 6.765, adjusted p = 0.023, partial η² = 0.116) in the Hbc compared to healthy controls, while MD and RD were unchanged (Table 2 and Fig. 2 a–d). In the total sample, HDRS score negatively correlated with FA ( r = -0.290, p = 0.016, adjusted p = 0.033, Fig. 2 e) and AD ( r = -0.402, p < 0.001, adjusted p = 0.003, Fig. 2 f) in the Hbc, after controlling for age, sex, and years of education. Within the MDD group, AD showed a significant negative correlation with HDRS score ( r = -0.394, p = 0.009, adjusted p = 0.036, Fig. S1 ). Illness duration was not associated with any DTI parameters in either the total sample or the MDD group. These findings indicate that MDD is associated with reduced white matter integrity and increased axonal injury in the Hbc, both of which are linked to depression severity. Table 2 Comparison of diffusion tensor imaging (DTI) parameters between patients with major depressive disorders and healthy controls. DTI parameters Control MDD MDD vs CON mean SE mean SE partial η² F (1,63) P-value P. Adjusted FA 0.206 0.007 0.184 0.005 0.129 6.997 0.010 0.023 AD 2.217 0.084 1.936 0.063 0.116 6.765 0.012 0.023 RD 1.571 0.080 1.509 0.060 0.007 0.401 0.548 0.547 MD 1.755 0.086 1.685 0.064 0.006 0.366 0.529 0.547 Estimated marginal means, standard error (SE), F- and P-values were obtained from one-way analysis of covariance (ANCOVA), with age, sex, and years of education included as covariates. The Benjamini-Hochberg procedure was applied for multiple comparisons. Significant group differences are presented in bold. MDD, patients with major depressive disorder; HC, healthy control participants. Chronic social defeat stress induces atrophy of the habenular commissure in Mice To investigate whether the alterations in Hbc DTI parameters observed in patients with MDD are recapitulated in an animal model, we employed a mouse model characterized by depressive-like behavior induced by CSDS. Using a 10-day CSDS paradigm (Fig. 3 a), mice exposed to CSDS exhibited significant behavioral deficits, including reduced social interaction time (SI, T (41) = 7.93, p < 0.001), lower SI ratio (T (41) = 2.22, p = 0.032), decreased time spent in the open arm of the EZM (T (41) = 2.98, p = 0.005), and increased immobility in the TST (T (41) = -3.34, p = 0.002) (Fig. 3 b–e). To investigate the relationship between behavioral impairments and Hbc structure, brains collected after behavioral testing were subsequently analyzed by light and electron microscopy (Fig. 4 a). Light microscopy revealed significant reduction in Hbc cross-sectional area in CSDS-exposed mice compared to naïve controls (T (18) = 3.59, p = 0.002, Fig. S2). Electron microscopy analysis showed significant reductions in axon diameter (T (18) = 5.76, p < 0.001, Fig. 4 c) and myelin thickness (T (18) = 6.70, p < 0.001, Fig. 4 d) in the Hbc of CSDS mice. Pearson’s correlation analyses revealed a significant positive relationship between social interaction time and the axon diameter of the Hbc ( r = 0.706, p < 0.001, adjusted p = 0.006, Fig. 4 b and e). A similar correlation was observed between social interaction time and myelin thickness ( r = 0.595, p = 0.006, adjusted p = 0.034, Fig. 4 b and f). Correlation analyses demonstrated that the degree of axonal constriction was associated with lower social interaction, linking structural atrophy to depressive-like behavior. These results suggest that chronic social stress induces atrophy of the Hbc, which may contribute to the development of depressive-like phenotypes in mice. Discussion To our knowledge, this study provides the first in vivo evidence that the Hbc exhibits microstructural abnormalities in MDD. Using DTI, we identified significant reductions in FA and AD in the Hbc of patients with MDD. Both measures were inversely associated with depressive symptom severity indexed by the HDRS, suggesting their potential as candidate biomarkers of disease severity. To validate these findings in a mechanistic model, we examined the Hbc in mice exposed to CSDS. Electron microscopy revealed reduced axon diameter and myelin thickness in the stressed group, and importantly, axonal constriction was correlated with impaired social interaction. These ultrastructural results support the interpretation that the abnormalities detected with DTI reflect genuine pathology of the Hbc. Taken together, convergent evidence from human and animal analyses highlights that structural compromise of the Hbc may contribute to the pathophysiology of depression and suggests that the Hbc may serve as a potential imaging biomarker. The LHb plays a pivotal role in processing negative-valence information, including negative reward prediction error and behavioral aversion, and exerts potent inhibitory influences on dopaminergic and serotonergic neurons in the midbrain through glutamatergic projections to GABAergic neurons in these regions 27 , 28 . Animal studies have demonstrated that LHb hyperactivity is associated with depressive-like behaviors, and that normalization of habenular firing ameliorates these phenotypes 11 , 29 . Consistent with these animal findings, a human fMRI study by Lawson and colleagues reported that unmedicated MDD patients exhibit abnormal task-related habenula responses during aversive conditioning, with reduced phasic activation to shock-associated cues, while healthy volunteers showed increased responses 30 . High-field fMRI further demonstrated that the LHb responds robustly to aversive or loss-related outcomes, supporting its role in negative reward processing 31 . These findings highlight the clinical relevance of habenular dysfunction. Structurally, the Hbc provides the sole commissural connection between the bilateral LHb. In vertebrate models, Horstick et al. demonstrated that motor asymmetry in zebrafish is mediated by habenular commissural projections and that disruption of this tract destabilizes left-right identity 32 . By extension, microstructural compromise of the Hbc in humans may impair coordination of bilateral habenular activity, producing asymmetric modulation of monoaminergic systems and amplifying mood dysregulation. At the cellular level, repeated stress may render the Hbc particularly vulnerable to degeneration. Elevated glucocorticoids impair oligodendrocyte proliferation and myelin maintenance 33 , 34 , while excessive glutamate release induces axonal and glial injury through excitotoxic mechanisms 35 , 36 . Oligodendrocytes express AMPA, NMDA, and metabotropic glutamate receptors, and overstimulation leads to Ca²⁺ influx and excitotoxic death 37 – 39 . Stress-induced inflammatory cytokines such as TNF-α and IL-6 further disrupt glutamate clearance by impairing glial transporters, exacerbating white matter injury 40 , 41 . These processes provide a plausible biological explanation for the reductions in FA, AD, and axonal diameter observed in the present study, consistent with axonal compromise. Although direct mechanistic evidence in humans remains limited, animal, cellular, and imaging data collectively suggest that stress-driven excitotoxic and inflammatory cascades may selectively compromise the integrity of the habenular commissure in MDD. In our cohort, FA and AD of the habenular commissure were significantly reduced in MDD, whereas RD and MD showed no differences. A decrease in AD is classically interpreted as a marker of axonal injury or reduced axonal caliber/coherence, supported by experimental models of traumatic or ischemic axonopathy where FA and AD decline together 42 – 45 . The absence of RD and MD alterations suggests that overt demyelination or global diffusivity changes were not detected. However, this should be interpreted cautiously given the small size of the Hbc and the limited spatial resolution of diffusion MRI, which may obscure subtle myelin pathology. Although EM analysis revealed reduced myelin sheath thickness in the CSDS model, RD did not differ between patients with MDD and healthy controls. This discrepancy likely reflects the limited specificity and spatial resolution of RD in detecting subtle or focal myelin changes in small tracts such as the Hbc. Differences in scale, tract size, and biological heterogeneity between preclinical and clinical samples may also contribute. Consistent with this possibility, ultrastructural analysis in CSDS mice revealed both reduced axon diameter and thinner myelin, supporting the notion that chronic social stress drives white-matter plasticity in stress-sensitive circuits, including the habenular pathways 46 , 47 . Taken together, these multiscale findings support a model in which axonal compromise of the Hbc constitutes a plausible microstructural substrate underlying depression pathophysiology. From a clinical standpoint, repeated DTI assessments of Hbc FA and AD could track MDD progression or treatment responsiveness. While medication effects cannot be entirely ruled out in the present study, further investigations involving medication-naïve patients or longitudinal cohorts could clarify whether Hbc abnormalities precede chronic depression or emerge as a secondary consequence. Moreover, neuromodulatory interventions targeting Hbc integrity–potentially through strategies that mitigate excitotoxicity or preserve oligodendrocyte function–could be explored as novel therapeutic approaches. Preserving commissural connectivity in the habenula may complement existing treatments aimed at normalizing LHb function. Notably, glutamate-based pharmacotherapies such as ketamine, an NMDA receptor antagonist, have demonstrated efficacy in ameliorating depression by modulating LHb activity 10 , 48 . Several limitations should be acknowledged. First, the cross-sectional design precludes causal inferences regarding the temporal relationship between Hbc alterations and the onset or progression of MDD. Second, most patients were receiving psychotropic medications, complicating the disentanglement of medication effects from disease-related changes in white matter integrity. Third, the small size and anatomical location of the Hbc pose challenges for conventional 3T diffusion MRI, making it susceptible to partial-volume artifacts. Future studies in longitudinal cohorts of medication-naïve patients with first-episode MDD as well as investigations employing higher-resolution imaging will be critical to determine whether Hbc abnormalities reflect trait vulnerability, state-dependent changes, or pathological scarring from recurrent MDD. In conclusion, this study integrates human DTI data with ultrastructural analyses in mice to highlight the potential significance of the Hbc in MDD. We observed reduced FA and AD in the Hbc, both of which differentiate patients from controls and correlate with depressive symptom severity, consistent with stress-related axonal compromise in rodent models. These findings position the Hbc as a tract-specific, translationally relevant marker. Further elucidation of its microstructural dynamics through longitudinal and high-resolution studies may inform the development of targeted interventions. These interventions could aim to preserve Hbc integrity, restore LHb function, and ultimately improve clinical outcomes in individuals with MDD. Declarations Competing Interest On behalf of all co-authors, the corresponding author declares that the authors have no competing financial or non-financial interests. Author contributions H.K., H.W.L., and K-M.H. conceived the study. K-M.H. and B-J.H. recruited the patients and conducted depression assessments. H.Y. and S.H.Y. performed animal experiments. W-S.T. performed the DTI analysis; H.Y. and W-S.T. carried out the statistical analyses. I.J.R., H-W.K., and E.Y. conducted electron microscopy experiments, and E.Y. analyzed the microscopic results. H.K. and K-M.H. supervised the study. H.W.L., H.Y., and H.K. wrote the manuscript. All authors reviewed and approved the final manuscript. 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Chronic social defeat stress caused region-specific oligodendrogenesis impairment in adolescent mice. Front Neurosci 2022; 16: 1074631. Bonnefil V, Dietz K, Amatruda M, Wentling M, Aubry AV, Dupree JL et al. Region-specific myelin differences define behavioral consequences of chronic social defeat stress in mice. Elife 2019; 8. Yang Y, Cui Y, Sang K, Dong Y, Ni Z, Ma S et al. Ketamine blocks bursting in the lateral habenula to rapidly relieve depression. Nature 2018; 554(7692): 317–322. Additional Declarations The authors have declared there is NO conflict of interest to disclose Supplementary Files YangetalSupplementaryInformation.docx Supplementary Information Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8207931","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":557802413,"identity":"98692110-e34c-4034-baec-e8d1e14b6cf9","order_by":0,"name":"Hyun Woo 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1","display":"","copyAsset":false,"role":"figure","size":5093417,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTractography and orientation distribution of the habenular commissure.\u003c/strong\u003e Tractography of the habenular commissure was generated using DSI studio, illustrating its connections between the left and right habenular nuclei. Both the habenular nuclei and the habenular commissure were clearly identified on the 3T MRI brain scan (a). The tractography of the habenular commissure (b) and the regions of interest (ROIs) corresponding to the habenular nuclei (c) are also shown. The orientation distribution function (ODF) of the habenular commissure, which characterizes the directional diffusion of white matter fibers, clearly demonstrates horizontally oriented, unidirectional diffusion within the commissure. Red arrows (d) indicate the ODF direction in each DTI voxel.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-8207931/v1/7c8e719073191c2ebe929a78.png"},{"id":98245366,"identity":"2f8ab27a-05ef-4111-b373-98edade7e594","added_by":"auto","created_at":"2025-12-15 16:17:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1703244,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAltered microstructural integrity of the habenular commissure in patients with MDD.\u003c/strong\u003e Group comparisons of diffusion tensor imaging (DTI) parameters in the habenular commissure (Hbc) between healthy controls (CON) and patients with MDD are shown. Compared with controls, patients with MDD exhibited significantly lower fractional anisotropy (FA; a) and axial diffusivity (AD; b), whereas no significant differences were observed in radial diffusivity (RD; c) or mean diffusivity (MD; d). Scatter plots show negative correlations between the Hamilton Depression Rating Scale (HDRS-21) scores and Hbc FA (e; \u003cem\u003er\u003c/em\u003e = -0.290, \u003cem\u003ep\u003c/em\u003e = 0.016) as well as Hbc AD (f; \u003cem\u003er\u003c/em\u003e = -0.402, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). (CON, \u003cem\u003en\u003c/em\u003e = 25; MDD, \u003cem\u003en\u003c/em\u003e= 43). Each dot represents an individual participant; error bars indicate the standard error of the mean (SEM). *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-8207931/v1/7a85027ecb0d45300613808f.png"},{"id":98245510,"identity":"5f2e6771-1f54-481a-97f9-3830382010bb","added_by":"auto","created_at":"2025-12-15 16:18:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1394612,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBehavioral alterations induced by chronic social defeat stress (CSDS).\u003c/strong\u003e (a) Schematic diagram of the experimental timeline. Mice were subjected to chronic social defeat stress for 10 consecutive days, consisting of daily physical defeat encounters followed by sensory stress exposure. Behavioral tests, including the social interaction (SI) test, elevated zero maze (EZM), and tail suspension test (TST), were conducted from day 11 to day 13, followed by tissue collection on day 14. (b and c) CSDS-exposed mice showed significantly reduced social interaction time and social interaction ratio compared with control (CON) mice in the SI test. (d) In the EZM, CSDS mice spent less time in the open arms, indicating increased anxiety-like behavior. (e) In the TST, CSDS mice displayed greater immobility time, reflecting enhanced depression-like behavior. Data are presented as mean ± SEM (CON, \u003cem\u003en\u003c/em\u003e = 20; CSDS, \u003cem\u003en\u003c/em\u003e= 23; *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; unpaired two-tailed \u003cem\u003et\u003c/em\u003e-test).\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-8207931/v1/7b5b2a0758c3742f9915b96e.png"},{"id":98245696,"identity":"f82f70ea-b6d3-4231-a847-2e56b47984aa","added_by":"auto","created_at":"2025-12-15 16:18:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4693630,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUltrastructural alterations of the habenular commissure in mice exposed to chronic social defeat stress (CSDS).\u003c/strong\u003e(a) Light microscopy image showing the location of the habenular commissure and representative transmission electron microscopy (TEM) images from control and CSDS mice. Red and blue outlines indicate myelin sheaths and axons, respectively. (b) Heatmap showing the Pearson’s correlation coefficients between behavioral metrics (social interaction [SI] time and ratio, elevated zero maze [EZM] time, and tail suspension test [TST] immobility) and axonal/myelination parameters (habenular commissure area, axon diameter, and myelin thickness). (c and d) Quantitative analyses showing significantly decreased axon diameter and myelin thickness in the habenular commissure of CSDS mice compared with controls. (e and f) Scatter plots showing positive correlations between SI time and both axon diameter and myelin thickness in the habenular commissure. Data are presented as mean ± SEM (CON, \u003cem\u003en\u003c/em\u003e = 8; CSDS, \u003cem\u003en\u003c/em\u003e= 12; *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; unpaired two-tailed \u003cem\u003et\u003c/em\u003e-test or Pearson’s correlation). Scale bars: 1 mm (a, top left), 100 µm (a, bottom left), 1 µm (a, top right and bottom right).\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-8207931/v1/9d33b3477a0b680c83c4c54c.png"},{"id":103506856,"identity":"cae149ab-d15a-4d35-ba2f-886b5cf2a484","added_by":"auto","created_at":"2026-02-26 13:39:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13004840,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8207931/v1/a8f5453a-a848-460d-a857-382615ee3e4e.pdf"},{"id":98245380,"identity":"a8c3a579-b561-4c9e-9c25-b2632254fe68","added_by":"auto","created_at":"2025-12-15 16:17:52","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1379770,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"YangetalSupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-8207931/v1/d3937ce75273352b7ebec200.docx"}],"financialInterests":"The authors have declared there is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose","formattedTitle":"Reduced commissural connectivity of the habenula in depression: Translational evidence from human DTI and rodent ultrastructural analyses","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMajor depressive disorder (MDD) is a prevalent psychiatric condition, affecting approximately 15\u0026minus;18% of individuals over their lifetime and is a leading contributor to global disease burden\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Core features of MDD include deficits in motivation and reward processing, and anhedonia is frequently observed in affected individuals\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Neurobiological evidence implicates dysfunction of the dopaminergic mesocorticolimbic pathway, which originates in the ventral tegmental area (VTA) and projects to regions including the prefrontal cortex, anterior cingulate cortex, and ventral striatum\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Neuroimaging studies have consistently identified structural and functional abnormalities within these circuits in MDD\u003csup\u003e\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eRecent work highlights the lateral habenula (LHb) as a critical regulator of midbrain dopaminergic and serotonergic centers. Hyperactivity of the LHb has been observed in animal models of depression, where it inhibits VTA dopamine neurons, potentially contributing to anhedonia-like behaviors\u003csup\u003e\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Despite increasing interest in the habenula, its commissural connection\u0026ndash;the habenular commissure (Hbc), a small white matter tract interconnecting the bilateral habenulae\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e\u0026ndash;remains largely unexplored in the context of MDD. Given that the LHb exerts bilateral control over the monoaminergic system, the integrity of the Hbc may represent a critical but overlooked factor in depressive pathophysiology.\u003c/p\u003e\u003cp\u003eDiffusion tensor imaging (DTI) allows non-invasive assessment of white matter microstructure and has revealed alterations in major tracts in MDD, including the corpus callosum\u003csup\u003e\u003cspan additionalcitationids=\"CR15 CR16 CR17\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. However, the Hbc has never been investigated in clinical populations, and thus its microstructural properties remain entirely uncharacterized. We focused on four standard DTI indices: Fractional anisotropy (FA) captures overall microstructural organization; axial diffusivity (AD) is particularly sensitive to axonal injury or reduced axonal caliber; radial diffusivity (RD) has been associated with myelin status; mean diffusivity (MD) reflects overall water mobility within tissue\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn this study, we examined whether the Hbc exhibits structural alterations in individuals with MDD. Using DTI, we quantified Hbc microstructure with standard indices of axona and myelin integrity. To further enhance microstructural resolution beyond the limits of clinical imaging, we complemented human DTI analyses with electron microscopy (EM) in a chronic social defeat stress (CSDS) mouse model. Through this integrative human-preclinical approach, we sought to determine whether Hbc microstructural abnormalities are associated with MDD and to evaluate their potential as a novel neuroimaging biomarker.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eEthics statement\u003c/h2\u003e\u003cp\u003e The procedures for animal experiments adhered to the guidelines of Korea University and were approved by the Korea University Institutional Animal Care and Use Committee (study approval number KOREA-2021-0073). Regarding the human neuroimaging study, the study protocol was approved by the Institutional Review Board (IRB) of Korea University Anam Hospital (2018AN0118). Written informed consent was obtained from all participants prior to their involvement in this study. The study methodology adheres to the approved guidelines and the principles outlined in the Declaration of Helsinki.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eParticipants\u003c/h3\u003e\n\u003cp\u003eA comprehensive cohort of 43 individuals diagnosed with MDD, comprising 31 females and 12 males, along with 25 healthy control participants, including 16 females and 9 males, was enrolled in the human brain imaging analysis. Recruitment of MDD patients occurred between July 2018 and March 2021 at the outpatient psychiatric clinic of Korea University Anam Hospital, Seoul, Republic of Korea. Inclusion criteria for MDD patients encompassed adults aged 19\u0026ndash;64 years, and the diagnosis was confirmed using the Structured Clinical Interview for the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5)\u003csup\u003e20\u003c/sup\u003e, conducted by two experienced board-certified psychiatrists (K.-M. Han and B.-J. Ham). Exclusion criteria included comorbid major psychiatric disorders (including personality and substance use disorders), MDD with psychotic features, acute suicidal or homicidal tendencies requiring inpatient treatment, history of serious or unstable medical illness, primary neurological conditions (e.g., cerebrovascular disease, Parkinson's disease, or epilepsy), recent abnormal results on physical examination or laboratory tests, pregnancy or nursing status, and any contraindications for magnetic resonance imaging (MRI), including claustrophobia.\u003c/p\u003e\u003cp\u003eTwenty-five healthy control participants aged 19\u0026ndash;64 years were recruited from the community through advertisement, following the same exclusion criteria that were applied to the MDD group by two psychiatrists. Healthy controls were included only if two board-certified psychiatrists confirmed the absence of any psychiatric history.\u003c/p\u003e\u003cp\u003eSociodemographic and clinical characteristics were systematically collected from both groups. The severity of depressive symptoms in all participants was evaluated by two psychiatrists using the 21-item Hamilton Depression Rating Scale (HDRS) at the time of the MRI scan\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. The duration of illness was assessed as the cumulative number of months with depressive episode(s) throughout the individual\u0026rsquo;s lifetime, employing life-chart methodology. Among the 43 MDD patients, 38 were taking psychotropic medication, including antidepressants or antipsychotics, at the time of study enrollment. Detailed information regarding psychotropic medications is presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDemographic and clinical characteristics for the participants\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCharacteristics\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMDD (n\u0026thinsp;=\u0026thinsp;43)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHC (n\u0026thinsp;=\u0026thinsp;25)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value (t, c\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAge\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e35.37\u0026thinsp;\u0026plusmn;\u0026thinsp;13.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e37.48\u0026thinsp;\u0026plusmn;\u0026thinsp;13.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.542 (t\u0026thinsp;=\u0026thinsp;0.613)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSex (Female/Male)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e31 / 12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e16 / 9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.486\u003c/p\u003e\u003cp\u003e(chi\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.485)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEducation years\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e13.98\u0026thinsp;\u0026plusmn;\u0026thinsp;2.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e16.80\u0026thinsp;\u0026plusmn;\u0026thinsp;1.96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001 (t\u0026thinsp;=\u0026thinsp;4.589)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHDRS-21 score\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e18.95\u0026thinsp;\u0026plusmn;\u0026thinsp;5.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001 (t\u0026thinsp;=\u0026thinsp;\u0026minus;20.667)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIllness duration (months)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e28.19\u0026thinsp;\u0026plusmn;\u0026thinsp;20.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDrug-naive / Drug-treated patients (n)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4 / 39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMedication, n\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAntidepressants\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSSRI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"9\" rowspan=\"10\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"9\" rowspan=\"10\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSNRI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNDRI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNaSSA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEtc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCombination of AD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAntipsychotics\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCombination of AP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003eData are presented as mean \u0026plusmn; standard deviation for age, years of education, HDRS-21 score, and illness duration. \u003cem\u003eP\u003c/em\u003e-values for sex distribution were obtained using the chi-square test. \u003cem\u003eP\u003c/em\u003e-values for comparisons of age, years of education, and HDRS-21 scores were obtained using an independent \u003cem\u003et\u003c/em\u003e-test. HCs, healthy controls; MDD, major depressive disorder; HDRS-21, 21-item Hamilton Depression Rating Scale; SSRI, selective serotonin reuptake inhibitor; NDRI, norepinephrine-dopamine reuptake inhibitor; SNRI, serotonin-norepinephrine reuptake inhibitor; NaSSA, noradrenergic and specific serotonergic antidepressant; Combination of AD, combination therapy with two or more antidepressants; APs, antipsychotics; Combination of AP, combination therapy with two or more antipsychotics.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eMRI acquisition\u003c/h3\u003e\n\u003cp\u003eParticipants underwent MRI-scanning at Korea University Anam Hospital using a 3.0 Tesla Siemens MR scanner (Magnetom Prisma, Siemens Medical Solutions, Erlangen, Germany) equipped with a 64-channel head and neck coil. A coronal 3D T1-weighted magnetization prepared rapid gradient echo (MPRAGE) sequence was acquired perpendicular to the long axis of the anterior commissure to the posterior commissure in the midsagittal plane, employing the following parameters: slice thickness (ST)\u0026thinsp;=\u0026thinsp;1.0 mm, no gap, number of slices\u0026thinsp;=\u0026thinsp;384, TR/TE\u0026thinsp;=\u0026thinsp;2300/2.26 ms, flip angle\u0026thinsp;=\u0026thinsp;9\u0026deg;, number of signal averages\u0026thinsp;=\u0026thinsp;1, matrix\u0026thinsp;=\u0026thinsp;256 \u0026times; 256, and field of view (FOV)\u0026thinsp;=\u0026thinsp;256 mm \u0026times; 256 mm. The acquisition of DTI data utilized a single-shot spin-echo planar imaging pulse sequence with the following specifications: number of diffusion gradient directions\u0026thinsp;=\u0026thinsp;64, matrix\u0026thinsp;=\u0026thinsp;112 \u0026times; 112, FOV\u0026thinsp;=\u0026thinsp;224 \u0026times; 224 mm\u003csup\u003e2\u003c/sup\u003e, slice thickness\u0026thinsp;=\u0026thinsp;2.0 mm, voxel size\u0026thinsp;=\u0026thinsp;2.0 \u0026times; 2.0 \u0026times; 2.0 mm\u003csup\u003e3\u003c/sup\u003e, echo time\u0026thinsp;=\u0026thinsp;55 ms, repetition time\u0026thinsp;=\u0026thinsp;6100 ms, b-value\u0026thinsp;=\u0026thinsp;1000 s/mm\u003csup\u003e2\u003c/sup\u003e, and flip angle\u0026thinsp;=\u0026thinsp;90\u0026deg;.\u003c/p\u003e\n\u003ch3\u003eDiffusion MRI preprocessing\u003c/h3\u003e\n\u003cp\u003eDiffusion preprocessing was performed using the FMRIB Software Library (FSL)\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Because the diffusion acquisition did not include reverse phase-encoding images, susceptibility-induced distortion correction using TOPUP could not be applied. Geometric distortions were minimized by using a relatively short echo time (TE\u0026thinsp;=\u0026thinsp;55 ms) and a posterior\u0026ndash;anterior phase-encoding direction, reducing susceptibility-related warping in deep midline structures such as the habenular commissure (Hbc). Eddy current and head-motion artifacts were corrected using the \u003cem\u003eeddy_cuda\u003c/em\u003e implementation, which also performs slice-wise outlier replacement and within-volume motion correction. All diffusion-weighted volumes were visually inspected to ensure geometric stability in the habenular region. Gradient nonlinearity correction and N4 bias-field correction were subsequently applied.\u003c/p\u003e\u003cp\u003eBilateral habenula regions of interest (ROIs) were manually delineated on each participant\u0026rsquo;s T1-weighted anatomical scan following established anatomical boundaries \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. T1 images were registered to diffusion space using affine transformation, and ROI alignment was visually verified for each participant.\u003c/p\u003e\n\u003ch3\u003eTractography and diffusion metrics\u003c/h3\u003e\n\u003cp\u003eDiffusion tensors were reconstructed in DSI Studio\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e using a standard tensor model, yielding voxel-wise FA, MD, RD, and AD maps. To visualize multidirectional fiber orientations within the Hbc, generalized q-sampling imaging (GQI) reconstruction was additionally applied to estimate the orientation distribution function (ODF).\u003c/p\u003e\u003cp\u003eDeterministic streamline tractography was conducted in DSI Studio. The left habenula served as the seed region, and the right habenula as the target. A QA-based anisotropy threshold was empirically determined based on prior habenula tractography studies. Tracking was performed with an angular threshold of 30\u0026deg;, a step size of 0.5\u0026ndash;1.5 voxels, and angular perturbation of 0\u0026ndash;95% to avoid local minima. Streamlines shorter than 5 mm or longer than 15 mm were excluded. For each participant, 100 streamlines were generated, and ODF was computed to characterize local fiber orientations.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eAnimals and chronic social defeat stress model\u003c/h2\u003e\u003cp\u003eMale C57BL/6J mice (Japan SLC, Inc., Shizuoka, Japan) and ICR mice (KOATECH, Korea) were obtained and housed in a facility under a 12-h light/dark cycle at 21\u0026ndash;24\u0026deg;C, with ad libitum access to food and water. The mice underwent a one-week habituation period to the housing conditions before being randomly assigned to either an experimental group or a control group (Na\u0026iuml;ve). Mice in the experimental group were exposed to chronic social defeat stress (CSDS) through daily 10-min encounters with ICR mice for 10 days, while control mice received no stress exposure and remained in their home cages. On the final day of the stress protocol, mice from both groups were immediately sacrificed following the last exposure. After decapitation, brains were rapidly extracted and processed for volumetric analysis.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eAnimal behavioral paradigms\u003c/h3\u003e\n\u003cp\u003eThe social interaction test consisted of two 150-s sessions: one with a social target (ICR mouse) and one without, as previously detailed\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. The test was conducted in an open-field arena (45 cm \u0026times; 45 cm \u0026times; 40 cm) containing a transparent box (10 cm \u0026times; 6.5 cm \u0026times; 20 cm) positioned in the interaction zone. During the first session, the box was empty, and during the second session, it contained the ICR mouse. Time spent in the interaction and non-interaction zones was quantified using EthoVision XT 12 (Noldus, Wageningen, Netherlands).\u003c/p\u003e\u003cp\u003eIn the elevated zero maze test, mice were placed in the closed quadrant at the beginning of the session and allowed to explore the maze freely for 5 min. Behavioral parameters, including total distance traveled, time spent in each quadrant, frequency, and latency to enter the open quadrant, were recorded and analyzed using EthoVision XT.\u003c/p\u003e\u003cp\u003eThe tail-suspension test was performed using a four-chamber apparatus separated by opaque acrylic partitions. Mice were suspended by the tail in each chamber using adhesive tape, and their behavior was recorded for 6 min. Immobility was quantified during the final 4 min of the session.\u003c/p\u003e\n\u003ch3\u003eTissue sampling and preparation\u003c/h3\u003e\n\u003cp\u003eMice were perfused transcardially with 0.9% saline followed by a fixative containing 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). Brains were removed and post-fixed overnight at 4\u0026deg;C in the same fixative. Coronal brain blocks (approximately 1.5 mm thick) containing the habenula commissure (Hbc) were prepared using a mouse brain matrix (Harvard Apparatus). The Hbc was dissected from each block and washed twice with 0.1 M phosphate buffer. It was then post-fixed in 1% osmium tetroxide for 90 min, dehydrated through an ascending ethanol series followed by propylene oxide, and embedded in Epon 812 (Oken Shoji, Japan).\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eElectron microscopic analysis of mouse Hbc\u003c/h2\u003e\u003cp\u003eFor electron microscopy, ultrathin sections (70 nm) were obtained from the middle portion of the Hbc using a Leica EM UC6 ultramicrotome (Leica Microsystems, Wetzlar, Germany). Sections were mounted on 200-mesh copper grids and examined with a Hitachi H-7650 transmission electron microscope (Hitachi, Tokyo, Japan) at 80 kV. Ten images were randomly acquired from selected regions within the Hbc at a magnification of 20,000\u0026times;. Images were analyzed using ImageJ by an investigator blinded to the experimental groups. Myelinated and unmyelinated axons were manually traced, and ImageJ was used to measure axon and myelinated axon diameters (Feret\u0026rsquo;s diameter, \u0026micro;m) and areas (\u0026micro;m\u003csup\u003e2\u003c/sup\u003e). Myelin thickness was defined as half the difference between the myelinated axon diameter and the axon diameter. Axon diameter distributions were characterized using an average of 127 axons per segment per control mouse and 178 axons per segment per CSDS mouse.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eFor the examination of human DTI data, multivariate analysis of covariance (MANCOVA) and one-way analysis of covariance (ANCOVA) were conducted. The four DTI parameters (FA, MD, RD, and AD) of the Hbc were treated as dependent variables, and groups (MDD vs healthy control) served as the independent variables. Age, sex, and years of education were included as nuisance covariates. In the supplementary analyses, potential correlations between illness burden-related clinical characteristics (severity of depressive symptoms [HDRS total score] and duration of illness) and DTI parameters were assessed using Pearson\u0026rsquo;s partial correlation. For analyses including the total sample, age, sex, and years of education were included as covariates. For analyses within the MDD group, illness duration and medication status (coded as 0 for drug-na\u0026iuml;ve patients; 1 for patients taking psychotropic medication) were additionally included. P-values were corrected using Benjamini-Hochberg procedure. To examine sociodemographic and clinical differences between the MDD and healthy control groups, independent \u003cem\u003et\u003c/em\u003e-tests were used for age, years of education, and HDRS scores, and chi-square tests were used for sex distribution. All statistical analyses were performed using R4.3.0.\u003c/p\u003e\u003cp\u003eComparisons of behavioral tests between CSDS and control mice were analyzed using two-tailed unpaired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test. Correlations between behavioral and electron microscopy parameters were assessed using Pearson\u0026rsquo;s \u003cem\u003er\u003c/em\u003e. Graphs were generated using the ggplot2 package.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eTractography of the habenular commissure\u003c/h2\u003e\u003cp\u003eUsing DSI Studio, we conducted an in-depth exploration of the habenular commissure, generating tractography results to depict the connections between the two habenular nuclei. The brain's 3T MRI scan provided a comprehensive view of both the individual habenular nuclei and the habenular commissure, serving as a reference for the subsequent analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and b). We closely examined specific ROIs associated with the habenular nuclei, emphasizing their significance in the overall analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). We also examined the orientation distribution function (ODF) of the habenular commissure, which describes the directionality of multidirectional diffusion in white matter regions. The ODF revealed a distinctive pattern of horizontal, unidirectional diffusion within the habenular commissure, providing insights into the microstructure of this neural pathway. To enhance clarity, red arrows were incorporated into the figure, indicating the direction of the ODF within the voxel of the diffusion tensor imaging (DTI). This comprehensive examination not only highlights the technical prowess of DSI Studio but also deepens our understanding of the habenular commissure's microstructural features, contributing to advancements in the field of neuroanatomical research (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eHuman DTI results\u003c/h2\u003e\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e summarizes demographic and clinical characteristics, including age, sex, years of education, HDRS score, illness duration, and psychotropic medications (i.e., antidepressants and antipsychotics). Age and sex did not differ between the groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.1), but the MDD group had fewer years of education than healthy controls (\u003cem\u003et\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.589, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Multivariate analysis of covariance revealed a significant main effect of group on Hbc DTI indices (Wilks\u0026rsquo; λ\u0026thinsp;=\u0026thinsp;0.670, Pillai\u0026rsquo;s trace\u0026thinsp;=\u0026thinsp;0.329, F\u003csub\u003e(4,60)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;7.36, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003cp\u003ePatients with MDD showed significantly reduced FA (F\u003csub\u003e(1, 63)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;6.997, adjusted \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.023, partial η\u0026sup2; = 0.129) and AD (F\u003csub\u003e(1, 63)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;6.765, adjusted \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.023, partial η\u0026sup2; = 0.116) in the Hbc compared to healthy controls, while MD and RD were unchanged (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea\u0026ndash;d). In the total sample, HDRS score negatively correlated with FA (\u003cem\u003er\u003c/em\u003e = -0.290, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.016, adjusted \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.033, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee) and AD (\u003cem\u003er\u003c/em\u003e = -0.402, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, adjusted \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef) in the Hbc, after controlling for age, sex, and years of education. Within the MDD group, AD showed a significant negative correlation with HDRS score (\u003cem\u003er\u003c/em\u003e = -0.394, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.009, adjusted \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.036, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Illness duration was not associated with any DTI parameters in either the total sample or the MDD group. These findings indicate that MDD is associated with reduced white matter integrity and increased axonal injury in the Hbc, both of which are linked to depression severity.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eComparison of diffusion tensor imaging (DTI) parameters between patients with major depressive disorders and healthy controls.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eDTI parameters\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003eMDD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c9\" namest=\"c6\"\u003e\u003cp\u003eMDD vs CON\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003emean\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSE\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003emean\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSE\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003epartial η\u0026sup2;\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eF\u003csub\u003e(1,63)\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eP-value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eP. Adjusted\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.206\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.007\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.184\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.005\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.129\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e6.997\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.010\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003e0.023\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2.217\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.084\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.936\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.063\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.116\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e6.765\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.012\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003e0.023\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.571\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.080\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.509\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.060\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.007\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.401\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.548\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.547\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.755\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.086\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.685\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.064\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.006\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0.366\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0.529\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0.547\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"9\"\u003eEstimated marginal means, standard error (SE), F- and P-values were obtained from one-way analysis of covariance (ANCOVA), with age, sex, and years of education included as covariates. The Benjamini-Hochberg procedure was applied for multiple comparisons. Significant group differences are presented in bold. MDD, patients with major depressive disorder; HC, healthy control participants.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eChronic social defeat stress induces atrophy of the habenular commissure in\u003c/h2\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003eMice\u003c/h2\u003e\u003cp\u003eTo investigate whether the alterations in Hbc DTI parameters observed in patients with MDD are recapitulated in an animal model, we employed a mouse model characterized by depressive-like behavior induced by CSDS. Using a 10-day CSDS paradigm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), mice exposed to CSDS exhibited significant behavioral deficits, including reduced social interaction time (SI, T\u003csub\u003e(41)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;7.93, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), lower SI ratio (T\u003csub\u003e(41)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.22, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.032), decreased time spent in the open arm of the EZM (T\u003csub\u003e(41)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.98, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.005), and increased immobility in the TST (T\u003csub\u003e(41)\u003c/sub\u003e = -3.34, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb\u0026ndash;e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo investigate the relationship between behavioral impairments and Hbc structure, brains collected after behavioral testing were subsequently analyzed by light and electron microscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Light microscopy revealed significant reduction in Hbc cross-sectional area in CSDS-exposed mice compared to na\u0026iuml;ve controls (T\u003csub\u003e(18)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3.59, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002, Fig. S2). Electron microscopy analysis showed significant reductions in axon diameter (T\u003csub\u003e(18)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;5.76, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec) and myelin thickness (T\u003csub\u003e(18)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;6.70, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed) in the Hbc of CSDS mice. Pearson\u0026rsquo;s correlation analyses revealed a significant positive relationship between social interaction time and the axon diameter of the Hbc (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.706, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, adjusted \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.006, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb and e). A similar correlation was observed between social interaction time and myelin thickness (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.595, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.006, adjusted \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.034, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb and f). Correlation analyses demonstrated that the degree of axonal constriction was associated with lower social interaction, linking structural atrophy to depressive-like behavior. These results suggest that chronic social stress induces atrophy of the Hbc, which may contribute to the development of depressive-like phenotypes in mice.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eTo our knowledge, this study provides the first \u003cem\u003ein vivo\u003c/em\u003e evidence that the Hbc exhibits microstructural abnormalities in MDD. Using DTI, we identified significant reductions in FA and AD in the Hbc of patients with MDD. Both measures were inversely associated with depressive symptom severity indexed by the HDRS, suggesting their potential as candidate biomarkers of disease severity. To validate these findings in a mechanistic model, we examined the Hbc in mice exposed to CSDS. Electron microscopy revealed reduced axon diameter and myelin thickness in the stressed group, and importantly, axonal constriction was correlated with impaired social interaction. These ultrastructural results support the interpretation that the abnormalities detected with DTI reflect genuine pathology of the Hbc. Taken together, convergent evidence from human and animal analyses highlights that structural compromise of the Hbc may contribute to the pathophysiology of depression and suggests that the Hbc may serve as a potential imaging biomarker.\u003c/p\u003e\u003cp\u003eThe LHb plays a pivotal role in processing negative-valence information, including negative reward prediction error and behavioral aversion, and exerts potent inhibitory influences on dopaminergic and serotonergic neurons in the midbrain through glutamatergic projections to GABAergic neurons in these regions\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Animal studies have demonstrated that LHb hyperactivity is associated with depressive-like behaviors, and that normalization of habenular firing ameliorates these phenotypes\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Consistent with these animal findings, a human fMRI study by Lawson and colleagues reported that unmedicated MDD patients exhibit abnormal task-related habenula responses during aversive conditioning, with reduced phasic activation to shock-associated cues, while healthy volunteers showed increased responses\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. High-field fMRI further demonstrated that the LHb responds robustly to aversive or loss-related outcomes, supporting its role in negative reward processing\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. These findings highlight the clinical relevance of habenular dysfunction. Structurally, the Hbc provides the sole commissural connection between the bilateral LHb. In vertebrate models, Horstick \u003cem\u003eet al.\u003c/em\u003e demonstrated that motor asymmetry in zebrafish is mediated by habenular commissural projections and that disruption of this tract destabilizes left-right identity\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. By extension, microstructural compromise of the Hbc in humans may impair coordination of bilateral habenular activity, producing asymmetric modulation of monoaminergic systems and amplifying mood dysregulation.\u003c/p\u003e\u003cp\u003eAt the cellular level, repeated stress may render the Hbc particularly vulnerable to degeneration. Elevated glucocorticoids impair oligodendrocyte proliferation and myelin maintenance\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, while excessive glutamate release induces axonal and glial injury through excitotoxic mechanisms\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Oligodendrocytes express AMPA, NMDA, and metabotropic glutamate receptors, and overstimulation leads to Ca\u0026sup2;⁺ influx and excitotoxic death\u003csup\u003e\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Stress-induced inflammatory cytokines such as TNF-α and IL-6 further disrupt glutamate clearance by impairing glial transporters, exacerbating white matter injury\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. These processes provide a plausible biological explanation for the reductions in FA, AD, and axonal diameter observed in the present study, consistent with axonal compromise. Although direct mechanistic evidence in humans remains limited, animal, cellular, and imaging data collectively suggest that stress-driven excitotoxic and inflammatory cascades may selectively compromise the integrity of the habenular commissure in MDD.\u003c/p\u003e\u003cp\u003eIn our cohort, FA and AD of the habenular commissure were significantly reduced in MDD, whereas RD and MD showed no differences. A decrease in AD is classically interpreted as a marker of axonal injury or reduced axonal caliber/coherence, supported by experimental models of traumatic or ischemic axonopathy where FA and AD decline together\u003csup\u003e\u003cspan additionalcitationids=\"CR43 CR44\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. The absence of RD and MD alterations suggests that overt demyelination or global diffusivity changes were not detected. However, this should be interpreted cautiously given the small size of the Hbc and the limited spatial resolution of diffusion MRI, which may obscure subtle myelin pathology. Although EM analysis revealed reduced myelin sheath thickness in the CSDS model, RD did not differ between patients with MDD and healthy controls. This discrepancy likely reflects the limited specificity and spatial resolution of RD in detecting subtle or focal myelin changes in small tracts such as the Hbc. Differences in scale, tract size, and biological heterogeneity between preclinical and clinical samples may also contribute. Consistent with this possibility, ultrastructural analysis in CSDS mice revealed both reduced axon diameter and thinner myelin, supporting the notion that chronic social stress drives white-matter plasticity in stress-sensitive circuits, including the habenular pathways\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Taken together, these multiscale findings support a model in which axonal compromise of the Hbc constitutes a plausible microstructural substrate underlying depression pathophysiology.\u003c/p\u003e\u003cp\u003eFrom a clinical standpoint, repeated DTI assessments of Hbc FA and AD could track MDD progression or treatment responsiveness. While medication effects cannot be entirely ruled out in the present study, further investigations involving medication-na\u0026iuml;ve patients or longitudinal cohorts could clarify whether Hbc abnormalities precede chronic depression or emerge as a secondary consequence. Moreover, neuromodulatory interventions targeting Hbc integrity\u0026ndash;potentially through strategies that mitigate excitotoxicity or preserve oligodendrocyte function\u0026ndash;could be explored as novel therapeutic approaches. Preserving commissural connectivity in the habenula may complement existing treatments aimed at normalizing LHb function. Notably, glutamate-based pharmacotherapies such as ketamine, an NMDA receptor antagonist, have demonstrated efficacy in ameliorating depression by modulating LHb activity\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eSeveral limitations should be acknowledged. First, the cross-sectional design precludes causal inferences regarding the temporal relationship between Hbc alterations and the onset or progression of MDD. Second, most patients were receiving psychotropic medications, complicating the disentanglement of medication effects from disease-related changes in white matter integrity. Third, the small size and anatomical location of the Hbc pose challenges for conventional 3T diffusion MRI, making it susceptible to partial-volume artifacts. Future studies in longitudinal cohorts of medication-na\u0026iuml;ve patients with first-episode MDD as well as investigations employing higher-resolution imaging will be critical to determine whether Hbc abnormalities reflect trait vulnerability, state-dependent changes, or pathological scarring from recurrent MDD.\u003c/p\u003e\u003cp\u003eIn conclusion, this study integrates human DTI data with ultrastructural analyses in mice to highlight the potential significance of the Hbc in MDD. We observed reduced FA and AD in the Hbc, both of which differentiate patients from controls and correlate with depressive symptom severity, consistent with stress-related axonal compromise in rodent models. These findings position the Hbc as a tract-specific, translationally relevant marker. Further elucidation of its microstructural dynamics through longitudinal and high-resolution studies may inform the development of targeted interventions. These interventions could aim to preserve Hbc integrity, restore LHb function, and ultimately improve clinical outcomes in individuals with MDD.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting Interest\u003c/h2\u003e\u003cp\u003eOn behalf of all co-authors, the corresponding author declares that the authors have no competing financial or non-financial interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e\u003cp\u003eH.K., H.W.L., and K-M.H. conceived the study. K-M.H. and B-J.H. recruited the patients and conducted depression assessments. H.Y. and S.H.Y. performed animal experiments. W-S.T. performed the DTI analysis; H.Y. and W-S.T. carried out the statistical analyses. I.J.R., H-W.K., and E.Y. conducted electron microscopy experiments, and E.Y. analyzed the microscopic results. H.K. and K-M.H. supervised the study. H.W.L., H.Y., and H.K. wrote the manuscript. All authors reviewed and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements:\u003c/h2\u003e\u003cp\u003eThis work was supported by the Brain Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT \u0026amp; Future Planning and by Korea University grant. This research was funded by Grant numbers RS-2023-00272290 (awarded to H.K) and NRF-2017R1D1A1B06032730 (awarded to H.W.L).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBromet E, Andrade LH, Hwang I, Sampson NA, Alonso J, de Girolamo G \u003cem\u003eet al.\u003c/em\u003e Cross-national epidemiology of DSM-IV major depressive episode. \u003cem\u003eBMC Med\u003c/em\u003e 2011; 9: 90.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGlobal Burden of Disease Study C. 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Here, we combined \u003cem\u003ein vivo\u003c/em\u003e human neuroimaging and preclinical ultrastructural analyses to identify a previously unrecognized microstructural signature of MDD. Using diffusion tensor imaging (DTI), we observed robust reductions in fractional anisotropy (FA) and axial diffusivity (AD) within the Hbc of individuals with MDD compared with healthy controls. Notably, FA and AD values were inversely correlated with Hamilton Depression Rating Scale (HDRS) scores, linking Hbc integrity to clinical symptom severity. Complementary electron microscopy of a chronic social defeat stress model revealed reduced axon diameter and myelin thickness of interhabenular connectivity, providing convergent evidence for commissural atrophy. These cross-species data identify the habenular commissure as a novel locus of microstructural pathology in depression and highlight its potential as a diagnostic and pathophysiological biomarker for MDD.\u003c/p\u003e","manuscriptTitle":"Reduced commissural connectivity of the habenula in depression: Translational evidence from human DTI and rodent ultrastructural analyses","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-15 16:09:29","doi":"10.21203/rs.3.rs-8207931/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"43356e38-5c3d-47f3-b6bb-5b04a96af251","owner":[],"postedDate":"December 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":59371255,"name":"Health sciences/Diseases/Psychiatric disorders/Depression"},{"id":59371256,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2026-02-24T14:27:20+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-15 16:09:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8207931","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8207931","identity":"rs-8207931","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}